牛型结核分枝杆菌MPT64蛋白的原核表达及纯化

Prokaryotic Expression and Purification of MPT64 Protein of Mycobacterium bovis

目的原核表达并纯化牛型结核分枝杆菌MPT64蛋白。方法以牛型结核分枝杆菌Vallee株全基因组DNA为模板,PCR扩增MPT64基因,克隆至pET-28a(+)载体,构建重组原核表达质粒pET-MPT64,转化至E.coli BL21(DE3)中,IPTG诱导表达。表达产物经SDS-PAGE鉴定后,用Ni-NTA亲和层析纯化,Western blot鉴定纯化蛋白。结果获得的MPT64基因测序结果与GenBank中登录的MPT64基因核苷酸序列同源性为100%;重组表达质粒pET-MPT64经酶切鉴定表明构建正确;表达的重组MPT64蛋白相对分子质量约为26 000,主要以可溶性形式表达,表达量约占全菌总蛋白的11%;纯化的重组MPT64蛋白纯度达93%,可与抗结核牛血清发生特异性反应。结论 成功原核表达并纯化了牛型结核分枝杆菌MPT64蛋白,其可作为诊断抗原用于新型牛结核病的检测。

Objective To express the MPT64 protein of Mycobacterium bovis in prokaryotic cells and purify the expressed product.Methods MPT64 gene was amplified by PCR using the whole genomic DNA of M.bovis Vallee strain as a template and cloned into vector pET-28a（＋）.The constructed recombinant plasmid pET-MPT64 was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed product was identified by SDS-PAGE,then purified by Ni-NTA affinity chromatography and further identified by Western blot.Results Sequencing result showed the homology of nucleotide of obtained MPT64 gene to that reported in GenBank was 100%.Restriction analysis proved that recombinant plasmid pET-MPT64 was constructed correctly.The expressed recombinant MPT64 protein,with a relative molecular mass of about 26 000,mainly existed in a soluble form and contained about 11% of total somatic protein.Purified recombinant MPT64 protein reached a purity of 93% and showed specific reaction with bovine serum against M.bovis.Conclusion The MPT64 protein of M.bovis was successfully expressed in prokaryotic cells and purified,which might be used as an antigen for diagnosis of novel bovine tuberculosis.